Unmodified natural phosphodiester oligonucleotides are cleaved by nucleases. Nuclease cleavage of these unmodified phosphodiester oligodeoxynucleotides is reported in serum and by introduction in other biological systems. Certain modifications to oligonucleotides have resulted in resistance to nucleases. One such modification that yields increased nuclease stability is the incorporation of methyl groups on the 2' position in an oligonucleotide. These nucleotides are prepared from the corresponding nucleosides and are usually introduced into the oligonucleotide using phosphoramidate intermediates. The usefulness of such 2'-O-modified oligonucleotides is such that certain 2'-O-nucleotide phosphoramidates are now commercially available from several sources both as the amidite and as the derivatized solid supports. These reagents are used to make oligonucleotides that in turn are used as diagnostic reagents, as research reagents and for other uses.
2'-O-Methyl ethers of ribonucleosides are natural modifications that are found in vivo as a minor component of many types of RNA. It has been shown that 2'-O-alkyl oligonucleotides are resistant to degradation by either DNA or RNA specific nucleases (Sproat, et. al., Nucleic Acids Res., 1989, 17, 3373), and form hybrids of high thermal stability with complementary RNA. Wagner., et. al., Nucleic Acids Research., 1991, 19, 5965-5971. These hybrids are useful as research reagents such as probes. Such probes allow for determining the functions of nucleic acids transcription factors and other related cellular entities. Such utility is illustrated in a number of publications as for example, Lamond, et. al., Cell, 1989, 58, 383-390 or Blencowe, et. al., cell, 1989, 59, 531-539 . As stated by Wagner, "2'-Methyloligoribo-nucleotides are oligonucleotide analogs which exhibit high resistance to both DNA and RNA specific nucleases and form hybrids of high thermal stability with complementary RNA." These analogs, as well as the recently described 2'-O-allyl oligoribonucleotides have proven to be valuable antisense compounds for studying snRNP-mediated pre-mRNA splicing and processing. Sequence-specific inhibition of histone pre-mRNA processing in vitro has been demonstrated using 2'-methyl or 2'O-ethyl oligoribonucleotide 19 mers complementary to the 5'-end of the U7-snRNP-RNA. These compounds were said to inhibit processing at a 300-fold lower concentration than that required using the corresponding DNA oligomer.
Synthesis of 2'-O-methyl oligoribonucleotides was first reported in 1985 using phosphodiester chemistry. Inoue, et. al., Nucleic Acids Res. Symposium Series 1985, 16, 165. Early methods of synthesizing 2'-O-methyl pyrimidine ribonucloesides generally started with the ribonucleoside. The ribonucleoside is protected at the primary 5'-hydroxyl. If necessary, the primary amino function of the aglycon was also protected. This is followed by a nonselective substitution of the 2'- and 3'-hydroxyls. This latter operation necessitates separation of the isomeric mixture by chromatography or fractional crystallization. Lengthy separations and low overall yields are a common limitation with this method of synthesis.
More recent methods of synthesizing 2'-O-methyl-pyrimidine ribonucleosides involve alkylation of 2'-O-methyl-1,3,5tri-O-benzoyl-e-D-ribose using diazomethane. The sugar used in the alkylation step is 1,3,5-tri-O-benzoyl-.alpha.-D-ribose. BF.sub.3 is conventionally used as a Lewis acid catalyst for this reaction. It has further been reported by C. Chavis,. st. al., J. Org. Chem., 1982, 47, 202-206, that BF.sub.3 etherate also prevent acyl migration during the synthesis. The product is purified by chromatography to give about a 75% yield. However, only small scale reactions (up to 5 g the product sugar) have been reported for this synthesis due to the hazards of using diazomethane which is an acutely toxic and explosive reagent.
There is a growing demand for large quantities of 2-O-methyl-1,3,5-O-benzoyl-.alpha.-D-ribose for use as a primary intermediate to synthesizing 2'-O-methyl-pyrimidine ribonucleosides. There is a further demand for large quantities of 2'-O-methyl-pyrimidine ribonucleosides for use in preparing oligonucleotides. There is a similar demand for large scale production of diazomethane, diazoethane and diazopropane.
It is an object of this invention to provide apparatus for a large scale, controlled generation of diazomethane, diazoethane and diazopropane.
It is a further object of this invention to provide improved processes for preparing 2-O-methyl-l,3,5-tri-O-benzoyl-.alpha.-D-ribose via a large scale diazomethane reaction along with the corresponding 2'-O-ethyl and 2'-O-propyl counterparts.